1

Cell Biology of Hyphae

INTRODUCTION

Members of the fungal kingdom are present in almost every conceivable niche. Even though fungi are remarkably diverse, many fungi share common cellular characteristics that are instrumental to the success of fungal growth, development, proliferation, and survival. The purpose of this introductory chapter is to provide the reader with an overview of some of the fundamental aspects of one of the predominant forms of fungal structureshyphae. The introductory chapter discusses the attributes that are common to many fungi as well as other organisms while also emphasizing some of the features that are unique to fungal species, as compared to other eukaryotes (some of the details will be discussed in depth in the following chapters of this book). Perhaps the primary recognizable difference between the hyphal cell and cells of other organisms is the predominantly coenocytic nature of the former. Characteristically, the hyphal cell harbors multiple nuclei that are evenly or unevenly distributed over relatively long distances of cytoplasmic continuity. Nonetheless, in this chapter, the term cell is used while discussing some of the fundamental as well as more unique attributes of hyphae. Identifying and understanding the nature of these attributes, and in particular, the regulatory mechanisms involved in orchestrating the growth of the fungal lament is an important step in the process of our intervention in fungal biology, be it curbing detriments or enhancing benets these organisms are capable of exhibiting.

described as an absorptive one. Thus, the accumulation of large concentrations of osmotically active molecules requires the presence of a structure that will assist in maintaining the integrity of the cell membranes. In addition, the adventurous, and at times, invasive proliferation of the fungal lament in a variety of diverse environments requires the presence of effective mechanisms to defend the fungal cell from external perils. The cell wall is a prime example of an efcient mechanical exocellular defense system. Understanding the structure of the cell wall and the synthesis of its components is essential for elucidation of the processes involved in fungal growth and development. This understanding includes the fundamental aspects of lament elongation and branching as well as various aspects of differentiation, pathogenicity, absorption, and secretion.

2.1

Composition

THE CELL WALL

The cell wall is a characteristic structure present in many organisms whose life style to grow in an environment with continuously changing water potential can be

The fungal cell wall accounts for approximately 25% of the mycelial dry weight. Approximately 80% of the cell wall dry weight is comprised of polysaccharides (Ruiz-Herrera 1992). The remaining 20% is comprised of proteins, lipids, and various inorganic salts. The predominant carbohydrate polymers found in different fungi are various forms of glucans and chitin. These sugars, synthesized and positioned in a nonuniform, yet highly regulated manner provide the external skeleton of the hyphal cell and are synthesized mainly at the apical region of the growing hyphae. Nonetheless, additional components (in particularcell wall-associated proteins) are involved in determining the cell surface properties of the growing or nascent hypha. Cell wall associated proteins are involved in the restriction of cell permeability to detrimental compounds and/or proteins present in the environment. These cell wall proteins are also

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involved in recognition of other fungi and regulation of processes such as anastomosis, sexual and asexual partner recognition, and interactions (e.g., recognition and adherence) with various hosts (Lora et al. 1995; Saupe et al. 1996). The interactions between fungal cell wall proteins and potential hosts is predominantly based on protein protein recognition, even though there are convincing records of carbohydrate protein interactions with the carbohydrate supplied by either the fungus or the host (Cormack et al. 1999).

in the Golgi apparatus is different than that described in mammals (Dean 1999). O-Glycosylation also occurs in lamentous fungi and is mediated by a conserved family of protein-mannosyl-transferases (Strahl-Bolsinger et al. 1999).

2.3

Hydrophobins

2.2

Synthesis

As the extension of the hyphal lament occurs mainly in the apical region, most of the biosynthetic machinery (and most likely some degradative machinery as well) involved in this process is trafcked to that region of the apical cell. As the main components of the hyphal cell are glucan and chitin, the glucan and chitin synthases must be positioned properly (in the vicinity of the extension or repair areas) and their activity regulated so as to produce the relevant polymer in a sitespecic manner. The mechanism by which the polymers are extruded is yet unclear. The presence of several chitin synthases in lamentous fungi has been documented at the respective genomic databases (NCBI and other, specic, databases as mentioned in Section 7.3 of this chapter). Based on the available information, Neurospora crassa and Aspergillus fumigatus each have seven genes encoding for chitin synthases, whereas Saccharomyces cerevisiae has three. The differential expression and functional consequence of chitin synthase gene inactivation have demonstrated that different chitin synthases have different roles during fungal growth and development (for a detailed perspective see Chapter 30). Some chitin synthases are essential for maintaining proper hyphal rigidity and form and are required for hyphal elongation. The specic roles of other members of this gene family have yet to be elucidated. The cell wall biosynthetic (chitin and glucan synthases) as well as cell wall lytic (chitinases and glucanases) enzymes required for cell elongation and branching processes are conveyed to the required location, and at least in some cases, compartmentalized trafcking is carried out in membranous vesicles (Sietsma et al. 1996). The wall at the tip is plastic allowing the extension of the cell by insertion of new material. As extension progresses, the material at the former position of the apex is progressively rigidied as it becomes the lateral wall of the growing cell. This rigidication is brought about by the covalent cross-linking of wall materials, especially chitin and b(1 . 3) glucans, and the hydrogen bonding of adjacent polysaccharide chains, especially chitin, to give microbrils. Many of the cell wall-associated proteins are heavily glycosylated. Many of the proteins secreted to the external face of the plasma membrane are linked via a remnant of the glycosylphosphatidylinositol anchor to the polysaccharide cell wall component. N-Glycosylation of proteins in the fungal endoplasmic reticulum is most likely very similar to that observed in mammalian cells, yet the process occurring
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Hydrophobins are among the unique protein components of the fungal cell wall. These small proteins, secreted during a variety of developmental processes are present in most lamentous fungi and have, so far, not been found in nonfungal species (Talbot 1999). These proteins play essential roles in fungal adherence, development of aerial structures, and infection of host plants by phytopathogens (Ebbole 1997). Fungal hydrophobins are hydrophobic in nature and can be dened by the presence of eight cysteine residues that are spaced in a particular manner within the amino acid sequence. They have been divided into two classes, based on solubility characteristics brought about by differences in cysteine residue spacing and distribution of hydrophobic and hydrophilic amino acids within the hydrophobin polypeptide sequences (Wosten et al. 1999). Beyond the involvement of fungal hydrophobins in natural processes of fungal growth and proliferation, the unique attributes of these proteins have been the basis of several suggested industrial applications. These include the use of hydrophobins as molecular anchor points for industrial proteins/enzymes, by attaching them to hydrophobic plastic surfaces. Other potential uses include the production of a natural protein coating over articial organs or other transplants (Kershaw and Talbot 1998). Though many structural components and organizational features are common to a wide range of fungi, differences in cell walls can be observed among different fungal species. Furthermore, the composition and structure of the cell wall can diverge immensely during the growth and development of the fungal colony.

THE PLASMA MEMBRANE

The presence of a cell wall provides the fungal cell with the ability to survive and grow without the need to equalize the cellular osmotic potential to that of its environment. In fact, the difference in osmotic potential contributes to the ability of the hyphal cell to elongate and branch by creating turgor pressure. The primary active barrier between the fungal cell and the environment is the plasma membrane. As seen in other organisms, the plasma membrane is a selective divide involved in ow of material and information between the cell and its environment. The maintenance of a plasma membrane potential and appropriate ion gradients are the basis of the ability to transport solutes in and out of the fungal cell. This ability is achieved via a variety of proton pumps, carrier proteins, and ion channels. N. crassa has been a prime model for analysis of

proton pumping and membrane potential (Davis 2000). The maintenance of a pH gradient and a high membrane potential (<200 mV, inside negative) is mainly dependent on the efux of protons, rather than Na (the common mechanism in animal cells). Nonetheless, even though the plasma membrane ATPase (a key element in the maintenance of the pH gradient) is essential, it is most likely that other processes contribute signicantly to the stabilization of cytoplasmic pH. These, most likely, include the modulation of the passive membrane permeability to protons, modulation of oxidative metabolism (a source of cytoplasmic protons), modulation of various proton-coupled transporters, and modulation of the ux between the cytosol and storage organelles (the vacuole in particular). A comprehensive overview of the transport processes operating in the fungal plasma membrane and the tonoplast has been compiled by Garrill (1994). Apart from the maintenance-related functions mentioned previously, the plasma membrane (and plasma membraneassociated proteins) plays a key role in additional biological functions of the hyphal cell. This includes serving as the anchoring structure for a variety of receptors involved in environmental sensing, a key location for initiation of endocytosis, and the nal step in secretion. Furthermore, all steps of hyphal fusion, be it self-fusion or fusion occurring with other strains during vegetative and/or mating processes involve recognition and changes in the plasma membrane.

SEPTATION

Compartmentalization of the fungal cell is dependent under most circumstances on the formation of septa. The initiation of septum formation has been associated with the recruitment of actin to a specic site along the hyphal cell. The events that follow include the deposition of cell wall material outside the plasma membrane. The septal wall provides a structural component for maintaining the hyphal architecture, yet is perforated in a way that provides the maintenance of cytoplasmic continuity and organelle movement (including, in some cases, nuclei) between compartments. Septation results in the cessation of growth and nuclear division in subapical cells. These subapical cells provide a reservoir of growth potential for the extending hypha. In most fungi, the nuclei residing within the subapical compartments ultimately give rise to new branches and various spore types. Thus, septation is most likely involved in determining both cell size and shape. In ascomycetes the septa generally appear to be homogeneous with a simple perforation while in basidiomycetes some septa are trilaminate. The primary septal plate carries at its center a thickened ring (the septal swelling) that surrounds the septal pore. Either face of the septal swelling is enclosed by the concave surface of a dome-shaped structure which is composed of a irregularly perforated multilayer membrane. This complex septal structure, termed the dolipore septum is characteristic of most basidiomycetes
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(excluding, for example, the rusts and the smuts). Occlusions within the pore and the parenthesomes on either side (in some fungi) delimit the pore domain (Moore 1985). The dolipore septum plays a role in nuclear migration, as the size of the pore and the parenthesome membrane provide a considerable barrier to nuclear movement. In fact, nuclear migration (as part of the dikaryon formation process) has been shown to be associated with disruption of the septum by lytic enzymes (Casselton and Economou 1985). Once the dikaryon is established, septal disruption no longer occurs. In contrast, it is important that septa remain intact for the maintenance of the binucleate stage. The cessation of septal disruption is probably one of the early postmating events regulated by the mating factors. One of the major risks imposed by cytoplasmic continuity characteristic of many lamentous fungi is the catastrophic event of massive leakage. Such events may occur following mechanical or chemical damage to the cell wall, which in severe instances can result in impairment of the lament structure to the point where cell contents can no longer be retained. A key mechanism in the process of minimizing the damage following the formation of a rupture in the fungal cell is the induction of rapid cell wall repair activities (RuizHerrera 1992) while at the same time activating a mechanism aimed at blocking the septal pores. One of the key factors involved in the latter process (in ascomycetes) is the Woronin body. Woronin bodies are proteinaceous granules that can reach a size of < 0.5 mm. They are of peroxisomal origin (Jedd and Chua 2000; Keller et al. 1991) and usually reside along the peripheral regions of the fungal cell and in association with the septum. The Woronin body is comprised of a crystalline subunit made of a protein with a peroxisometargeting signal that most likely indicates that these bodies originate from the peroxisomal membrane. Even though the Woronin bodies do not move along with normal cytoplasmic streaming, the dramatic increase in cytoplasmic ow following cell rupture may provide sufcient force to pry the Woronin bodies from their plasma membrane site of attachment, and provide the vehicle for a rapid direction of the bodies to the septal pore.

THE CYTOSKELETON

The hyphal cytoskeleton plays a key role in cell shape determination, maintenance, and growth. A broad spectrum of proteins interacts with the basic components of the hyphal cytoskeleton that is comprised of microtubules and actin laments. The dynamics of the hyphal cytoskeleton that involves constant change and adjustment in concert with cellular changes is characteristic of lamentous fungi. Furthermore, it is most likely that cytoskeletal elements interact directly with components of the cell wall. Thus, structure, sensing, and cellular response are closely associated with the hyphal cytoskeleton. In contrast to many other cells types (including yeasts and higher eukaryotic cells) the distances that some cellular

components must travel within the elongated hyphal cell are enormous. The continuous supply of vesicles (produced in the Golgi apparatus) to the apical cluster is critical for tip growth. This cluster of vesicles also termed the Spitzenkorper (apical body) appears as a dense spherical body with no discrete outline and is associated only with growing hyphae (apical cells) or immediately prior to the formation of new branches. The vesicles within the apical body range in diameter from 30 to 400 nm. The content of these vesicles, which most likely includes (among other proteins) enzymes involved in cell wall biosynthesis, has yet to be properly determined. Perhaps the most studied components of the apical body are a subpopulation of the smaller vesiclesthe chitosomes. The pioneering work of Braker et al. (1976) established the linkage between these vesicles and chitin synthase activity. Following the isolation of genes encoding chitin synthases and the availability of antichitin synthase antibodies, localization of chitin synthases to chitosomes by immunohystochemistry was made possible (Sietsma et al. 1996). The capacity to transfer cargo along the cytoskeletal backbone of the hyphal cell is provided by molecular motors that are responsible for both forward and retrograde transport along the hyphal lament. The proteins that provide the ATP-driven motor function along actin and microtubules are myosin and kinesin/dynein, respectively (see later). Recent observations by Seiler et al. (1999) suggest that signicant retrograde transport, at times exceeding the forward trafc of vesicles occurs in the vicinity of the hyphal tip. Thus, transport of compounds absorbed from the environment, or recycling of cellular proteins and other components from the hyphal tip region are also key processes involved in hyphal elongation. The efcacy of vesicle transport is much dependent on the proper function of nascent and mobile cytoskeletal elements. Actin microlaments form longitudinally oriented cables and ne laments in the cytoplasm extending, in some cases, to the apical dome and they are also associated with the Spitzenkorper. Microtubules are longitudinally oriented and they do not usually extend into the apical dome. Interestingly, not every case of functional disruption of cytoskeletal elements (by genetic alteration or drug treatment) results in measurable impairment of vesicle trafc or hyphal tip growth (Yamashita and May 1998). Furthermore, it appears that the organization of microtubules, as are the cytoskeletal motor complexes may well be diverse in different lamentous fungal species (and, apart from the structural conservation of the core elements, are also functionally diverse from yeasts). Microtubules are involved in the formation of the mitotic spindle, intracellular transport of secretory vesicles (Howard 1981), positioning of the Spitzenkorper (Riquel et al. 1998), and organelle movement (Steinberg and Schliwa 1993). The initiation of microtubule polymerization (which involves the structurally-related a and b tubulin proteins) is determined by spindle pole bodies (microtubule organization centers) (Heath 1994). In vegetative hyphae, another tubulin, designated g-tubulin (which is slightly less structurally similar to a and b tubulin than they are to each other) is present only in the
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spindle pole bodies. These spindle pole bodies are a critical determinant in the establishment of microtubule polarity and the concomitant direction of cargo transport within the hyphal cell. Kinesins: The presence of members of the kinesin superfamily (comprised of the founder family of conventional kinesins along with kinesin-like proteins) in fungi is well documented. Lee and Plamann (2001) have described the structural and functional attributes of fungal kinesins (and other cytoskeletal components). The role of conventional and kinesin-like proteins in vesicle transport towards the hyphal tip as well as mitochondrial and nuclear movement have been demonstrated, yet their function in different lamentous fungi may vary (Lee and Plamann 2001; Steinberg and Schliwa 1995; 1996). Dynein: Dynein and its accompanying multisubunit component, known as dynactin have been implicated to be involved in vesicle trafcking and mitosis. The multisubunit dynein/dynactin motor is complex and all indications are that their structure is highly similar to those present in higher eukaryotes. The complex is involved in retrograde transport, vesicle movement associated with the endocytic pathway, formation of the endoplasmic reticulum network, organization of the Golgi apparatus, and formation of the mitotic spindle (Karki and Holzbaur 1999). Plamann et al. (1994) have analyzed the abnormalities in nuclear distribution associated with defects in cytoplasmic dynein and have proposed models for the possible mechanisms by which the complex is involved in nuclear migration.

REGULATION OF ELONGATION/ BRANCHING

Hyphal elongation and branching are fundamental processes that dene the morphology of lamentous fungi. The efcacy and success of these processes are essential for the outreach and acquisition of nutrients from the environment and in many cases, are part of the proliferative and reproductive cycle. The formation of the common cylindrical cell extension/branches by the polarized synthesis of new membrane and cell wall material must be both temporally and spatially regulated. Though information concerning the regulation of hyphal growth is accumulating, this aspect of hyphal cell biology is still in its infancy and a comprehensive picture of the network of processes involved has yet to be obtained. The use of genetics has been instrumental in identifying and analyzing factors involved in the regulation of polar growth in both yeasts and lamentous fungi. The signicant morphological differences between yeast and lamentous fungi suggest that in addition to the common basic machinery involved in establishment, maintenance, and changes in cell polarity, lamentous fungi may have additional or alternate regulatory pathways and are most likely to have more downstream elements (as required by their more complex

morphology) governed by the regulatory systems involved. A case illustrating a signicant difference in a key regulatory pathway involved in polar growth of yeasts and lamentous fungi is described later. Mutants that exhibit abnormal morphology of hyphal growth have served for identifying some of the genes involved in polar extension/branching of hyphae and the linkage between cell polarity and hyphal extension. Bruno et al. (1996) analyzed a temperature-sensitive Neurospora mutant (mcb) that exhibited apolar hyphal growth, which was not restricted to the apical cell of the hyphal lament. The mutant was shown to be defective in the gene encoding the regulatory subunit of cAMP-dependent protein kinase (PKA). The fact that the PKA pathway is involved in the regulation of polarized hyphal growth (the mutant is also defective in septum localization) in N. crassa is consistent with reports on the involvement of an activated PKA pathway in developmental switches that affect growth polarity in Ustilago maydis and Magnaporthe grisea (lamentous growth and proper appresorium formation, respectively). Later events in the penetration and infection processes of these fungi are stimulated by a mitogen-activated protein (MAP) kinase cascade, presumably through a MAP kinase module that may respond to the cAMP signal (see Chapter 4). In contrast, even though an increase in PKA activity in S. cerevisiae has morphological consequences, these do not really involve cell polarity. This difference may reect the different nature of growth of lamentous fungi, in which septation/cell separation is not linked with polar elongation (yet involves cross-talk between mechanisms regulating cell size and cell cycle). The comprehensive mechanistic involvement of PKA in regulation of polar growth and other cellular processes has yet to be obtained; most likely it involves the complex regulation of PKA expression itself, the interaction between PKA and its substrates, and interactions between PKA and other regulatory pathways. A variety of hyperbranching mutants have been obtained in different lamentous fungi. The genetic dissection of these mutants is at its infancy. An example of an immediate need to understand such a mutant was the appearance of a hyperbranched colonial mutant of Fusarium graminearum strain A3/5, used to produce Quornw myco-protein. The unexpected increase in the population of these colonial mutants in continuous ow cultures suggested a selective advantage over the sparsely branched wild type strain. The apparent advantage is an altered capability of glucose metabolism. Thus, the hyperbranching phenotype is due to a pleiotropic effect of the mutation in the colonial strain (Trinci et al. 1999). The inuence of nutrient sources and the availability it has on colony morphology, as well as the effects paramorphogens have on fungal growth, and the means available to study the growth of the fungal colony have been recently reviewed by Olsson (2001). The analysis of Neurospora colonial temperature sensitive (cot) mutants has yielded information concerning elongation and branching. Close examination of these mutants has claried the fact that not all hyperbranching events are
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identical in rate of the elongation/branching frequencies or morphology of the branching pattern. Thus, it has become clear that diverse factors can result in considerably similar alterations in gross morphology while the mechanisms involved in imposing these changes can be very diverse, thus requiring much additional investigation. At the permissive temperature, cot-1 grows in a manner almost indistinguishable from that of the wild type, yet when shifted to the restrictive temperature hyphal elongation ceases concomitant with a massive induction of hyphal branching (Collinge et al. 1978; Yarden et al. 1992). The newly formed hyphal tips are unable to continue elongating at the restrictive-temperature, but returning the culture to the permissive-temperature results in the rapid restoration of normal hyphal growth. The cot-1 gene has been cloned and, based on the deduced COT1 amino acid sequence it encodes a Ser/Thr-specic protein kinase that can be membrane associated (Gorovits et al. 1999; Yarden et al. 1992). Furthermore, evidence for a linkage between branching and fungal cytoskeleton assembly and function (Plamann et al. 1994) has set the stage for further analysis of the regulation and interaction of components governing the polarity of fungal lament growth. Ser/Thr kinases that are highly similar to COT1 have been identied in other fungi (Buhr et al. 1996; Verde et al. 1998) and many higher eukaryotes, including Candida elegans, Drosophila, and mammals (Gorovits et al. 1999). The COT1 is highly similar to the product of the human DM kinase gene, which when mutated can cause myotonic dystrophy (Mahadevan et al. 1993). The common feature of most cells with alterations in their COT1-related kinase is abnormal morphology and observable defects in their polarity. In N. crassa, COT1 kinase is essential, as insertional inactivation of the cot-1 gene is lethal (Yarden et al. 1992). Lauter et al. (1998) have demonstrated that cot-1 transcription is photoregulated. Using co-imunoprecipitation with antiCOT1 antibodies, Gorovits et al. (1999) have suggested the feasibility of a physical interaction between COT1 kinase and the catalytic subunit of type 2B phosphatase (calcineurin). Interestingly, a reduction in calcineurin activity (via antisense expression of the cna-1 gene) also results in reduced hyphal elongation rate accompanied by hyperbranching (Prokisch et al. 1997). It is not surprising that different kinases/phosphatases are involved in hyphal elongation/ branching, as they are involved in almost every cellular process. If and how the various components of different signal transduction pathways interact in lamentous fungi is far from being fully understood. The regulation of hyphal elongation/branching is probably complex, and involves a variety of signal transduction pathways. In addition to these pathways, it is conceivable that master regulators of gene expression involved in hyphal growth are involved. One such example may be the B genes in U. maydis (Brachmann et al. 2001). Another potential regulator of hyphal branching is the pah1 homeobox gene of Podospora anserina (Arnaise et al. 2001).

Overall, even with the abundance of colonial mutants isolated from a variety of lamentous fungi, the understanding of the genetic basis of these morphological abnormalities remains scarce (Turner and Harris 1999). Nonetheless, the availability of so many mutants may well prove an asset for expansion of the current attempts to functionally analyze genes involved in hyphal elongation and branching. Elucidation of this process is important not only for satisfying the basic need of understanding such a fundamental process, but also as a starting point for rationally intervening in this process. Curbing the proliferation of pathogens, or enhancing growth and morphogenesis in industrial strains, would both benet once a clearer mechanistic picture of these events is obtained. Much hope has been put on the expected increase in secretion from hyperbranching mutants. The components involved in secretion in lamentous fungi are highly similar to those found in yeasts and higher eukaryotes. However, in contrast to other organisms, the hyphal cell with its polar apical extension creates a unique morphological arena for the process of secretion. Much focus has been put on understanding secretion at the apical zone of growing hyphae (Conesa et al. 2001). However, even though a link was proposed between growth and secretion at hyphal tips in lamentous fungi (Wessels 1993), the increase in hyphal tips per unit of biomass does not necessarily result in a parallel increase in secretion of extracellular enzymes (Conesa et al. 2001; Trinci et al. 1999).

solid foundation for resolving many fundamental questions in fungal biology.

7.2

Cell Imaging

7 7.1

STUDYING THE BIOLOGY OF THE HYPHAL CELL Biochemical Genetics

A lamentous fungus was used to open the door to biochemical genetics (Beadle and Tatum 1941). Since that bridge between genes and their biochemical function was formed, the biochemical genetics approach has become part and parcel of almost every analytical venture in biology. Thus, understanding biochemical pathways and the genetic basis of the structural and regulatory elements from which they are comprised is still a common and efcient strategy to advance our understanding of any cellular process. The haploid nature of many fungi (during most of their life cycle), the availability of mutants (or the means to produce them) and the development of methodology by which almost any fungal species can be made amenable to DNA-mediated manipulations provide a likely assurance that biochemical genetics approach, along with others, will continue to be in the mainstream of fungal biology research. The availability of complete fungal genomes has become a reality (see later) and the high-throughput technology encompassing the genomics and postgenomics era are certain to contribute immensely to the future advances in understanding fungal biology. Nonetheless, the power of classical genetics linked to biochemistry has not diminished and will most likely continue to serve as a
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Macroscopic and microscopic observation has always been a key element in the analysis of the fungal cell. Thus, as early as the third quarter of the 17th century Sir Robert Hooke, utilizing his optical magnication apparatus described the fungal cell (amongst those of other organisms he observed). Microscopy has accompanied the mycologist ever since and still provides an important means in studying fungal biology. The availability of reporter systems along with molecular transformation techniques and recent advances (as well as traditional technologies) in microscopy (light, uorescent, confocal, and electron) allows the detailed probing of events at the cellular level. Measuring the relative intracellular ion concentrations in specic areas of hyphae (and other fungal structures) is possible by X-ray microanalysis procedures (Connolly et al. 1999). Immunocytochemical techniques (utilizing uorescent or gold particle-labeled antibodies) are accessible and have been integrated into a growing number of study systems for localization studies, colorimetric visualization, and measurement of reporter gene expression in xed or live tissues (e.g., b-glucoronidase or green uorescence protein systems). Perhaps the most exciting current developments are the improvements in live fungal cell imaging. The use of classic dyes that are sensitive to changes within the living cell (e.g., pH, specic ions, reactive oxygen species, etc.) along with dyes that are membrane selective (e.g., FM4-64) and in combination with uorescent and confocal microscopy will certainly open new avenues of research that will shed light on the cellular biology of the hyphal cell (Fischer-Parton et al. 2000; Heath 2000).

7.3

High-Throughput Analyses (The X-omics Age)

The rst fungal genome to be sequenced in its entirety was that of S. cerevisiae in 1997. Since then, several fungal genomes have been sequenced (some more than once) by private ventures. Recently, the rst complete sequence of a lamentous fungus, N. crassa, has been made available to the public (http://www-genome.wi.mit.edu/annotation/fungi/ neurospora/). The Aspergillus nidulans genome is also available, with restrictions (http://www.fgsc.net/ aspergenome.htm). Others are likely to follow soon, as public efforts for sequencing the genomes of several additional lamentous fungi are under way. These include Cryptococcus neoformans (http://www-sequence.stanford.edu/group/C. neoformans/), M. grisea (http://www.riceblast.org/), A. fumigatus (http://www.sanger.ac.uk/ Projects/A_fumigatus/), Candida albicans (http://sequence-www.stanford.edu/ group/candida/index.html), Phanerochaete chrysoporium

(http://www.jgi.doe.gov/ programs/whiterot.htm), and others. The sequencing of entire genomes will provide the required information for enhancing the ability to perform highthroughput analyses of gene structure and function (Sweigard and Ebbole 2001). The age of structural and functional genomics, transcriptomics, proteomics, metabolomics, and additional categories and subcategories has begun. The prospects for utilizing the new data and technological tools for understanding the molecular basis of cellular events in the hyphal cell are enormous (see Chapters 12 and 17). It will now be easier to combine reductionist approaches (e.g., analysis of single gene/protein function) with some of the more holistic approaches involving the concurrent study of several components affecting a specic trait.

missing links in conserved pathways, while at the same time point to unique proteins in fungal species. Nonetheless, at the end of the day, classical conceptual approaches used in cell biology research during the past decades will be required in order to answer the fundamental questions relating to growth and development of the hyphal cell.

ACKNOWLEDGEMENTS
The author thanks Hans Sietsma and Talma Katan for their critical comments on this manuscript and the Israeli Academy of Sciences for its continuous support in studying the hyphal cell.

CONCLUSIONS

REFERENCES
Arnaise S, Zickler D, Poisier C, and Debuchy R (2001). pah1: a homeobox gene involved in hyphal morphology and microconidiogenesis in the lamentous ascomycete Podospora anserina. Mol Microbiol 39:54 64. Beadle GW and Tatum EL (1941). Genetic control of biochemical reactions in Neurospora. Proc Natl Acad Sci USA 27:499 506. Brachmann A, Weinzierl G, Kamper J, and Kahmann R (2001). Identication of genes in the bW/bE regulatory cascade in Ustilago maydis. Mol Microbiol 42:1047 1063. Braker CE, Ruiz-Herrera J, and Bartnicki-Garcia S (1976). Structure and transformation of chitin synthase particles (chitosomes) during microbril synthesis in vitro. Proc Natl Acad Sci USA 73:45704574. Bruno KS, Aramayo R, Minke PF, Metzenberg RL, and Plamann M (1996). Loss of growth polarity and mislocalization of septa in a Neurospora mutant altered in the regulatory subunit of camp-dependent protein kinase. EMBO J 15:5772 5782. Buhr TL, Oved S, Truesdell GM, Huang C, Yarden O, and Dickman MB (1996). TB3, a glutamine-rich kinase-encoding gene from Colletotrichum trifolii complements the cot-1 mutant of Neurospora crassa. Mol Gen Genet 251:565 572. Casselton LA and Economou A (1985). Dikaryon formation. In: Moore D, Casselton LA, Wood DA, Frankland JC eds. Developmental Biology of Higher Fungi. Cambridge: Cambridge University Press. pp 213 229. Collinge AJ, Fletcher MH, and Trinci APJ (1978). Physiological and cytology of septation and branching in a temperature-sensitive colonial mutant (cot-1) of Neurospora crassa. Trans Br Mycol Soc 71:107 120. Conesa A, Punt P, van Luik N, and van den Hondel CAMJJ (2001). The secretion pathway in lamentous fungi: a biotechnological view. Fungal Genet Biol 33:155 171. Connolly MS, Williams N, Heckman CA, and Morris PF (1999). Soybean isoavones trigger a calcium inux in Phytophthora soja. Fungal Genet Biol 28:6 11. Cormack BP, Ghori N, and Falkow S (1999). An adhesin of the yeast pathogen Candida glabrata mediating adherence to human epithelial cells. Science 285:578 582. Davis RH (2000). Neurospora, contributions of a model organism. Oxford: Oxford University Press. pp 209 259. Dean N (1999). Asparagine-linked glycosylation in the yeast Golgi. Biochim Biophys Acta 1426:309 322.

Most fungi posses most of the common cellular features shared by other eukaryotes. However, the distinct shape and some of the metabolic features of the hyphal cell require that the activities of the fundamental, conserved, cellular machinery be amended with the unique structural and functional elements required to maintain the hyphal cell and support its growth. Thus, identifying the structural components (common and unique), determining their function and elucidating the regulatory mechanisms responsible for the concerted interactions between subcellular components on the one hand and the intact organism and its environment, on the other, are instrumental for our efforts to intervene with the activities of these fascinating and diverse organisms. The advances made in dissection of individual components of the hyphal cell (e.g., cell wall, organelles, and septa) are signicant. The combination of biochemistry, genetics (classical and molecular), and microscopy have yielded impressive results in the process of identifying many of the components involved, determining their signicance in hyphal growth, and associating them with the topography of the hyphal cell. However, we are still challenged with a need to understand many of the mechanistic aspects of hyphal cell biology. At times, the difculties involved in the analysis of these aspects are shared by those studying other eukaryotic cells (e.g., function of membrane-associated proteins, organization and function of cytoskeletal elements, and regulation of the cell cycle, etc.) and conceptual or technological breakthroughs in the eld of cell biology are likely to be of signicant value to fungal biologists. However, understanding the unique aspects linked to fungal morphology and growth, and intervening in these processes, depend on advances in mycological research in general and the in-depth analysis of fungal species of interest. The outcome of the current effort invested by members of the fungal biology community in advancing the elds of fungal genomics and proteomics may well prove to be an invaluable stepping-stone in better understanding the hyphal cell. Comparative genomics (among lamentous fungi and between fungi and other organisms) will assist in identifying
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Ebbole DJ (1997). Hydrophobins and fungal infection of plants and animals. Trends Microbiol 5:405 408. Fischer-Parton S, Parton RM, Hickey PC, Dijksterhuis J, Atkinson HA, and Read ND (2000). Confocal microscopy of FM4-64 as a tool for analysing endocytosis and vesicle trafcking in living fungal hyphae. J Microsc 198:246 259. Garrill A (1994). Transport. In: Gow NAR, Gad GM eds. The Growing Fungus. London: Chapman & Hall. pp 163 181. Gorovits R, Propheta O, Kolot M, Dombradi V, and Yarden O (1999). A mutation within the catalytic domain of COT1 kinase confers changes in the presence of two COT1 isoforms and in Ser/Thr protein kinase and phosphatase activities in Neurospora crassa. Fungal Genet Biol 27:264 274. Heath IB (1994). The cytoskeleton. In: Gow NAR, Gad GM eds. The Growing Fungus. London: Chapman & Hall. pp 99 134. Heath MC (2000). Advances in imaging the cell biology of plant microbe interactions. Ann Rev Phytopathol 38:443 459. Howard RJ (1981). Ultrastructural analysis of hyphal tip cell growth in fungi: Spitzenkorper, cytoskeleton and endomembranes after freeze-substitution. J Cell Sci 48:89 103. Jedd G and Chua NH (2000). A new self-assembled peroxisomal vesicle required for efcient resealing of the plasma membrane. Nat Cell Biol 2:226 231. Karki S and Holzbaur ELF (1999). Cytoplasmic dynein and dynactin in cell division and intracellular transport. Curr Opin Cell Biol 11:45 53. Keller GA, Krisans S, Gould SJ, Sommer JM, Wang CC, Schliebs W, Kunau W, Brody S, and Subramani S (1991). Evolutionary conservation of a microbody targeting signal that targets proteins to peroxisomes, glyoxysomes, and glycosomes. J Cell Biol 114:893 904. Kershaw MJ and Talbot NJ (1998). Hydrophobins and repellants: proteins with fundamental roles in fungal morphogenesis. Fungal Genet Biol 23:18 33. Lauter F-R, Marchfelder U, Russo VEA, Yamashiro CT, Yatzkan E, and Yarden O (1998). Photoregulation of cot-1, a kinaseencoding gene involved in hyphal growth in Neurospora crassa. Fungal Genet Biol 23:300 310. Lee IH and Plamann M (2001). Microtubules and molecular motors. In: Howard RJ, Gow NAR eds. The Mycota VIII. Biology of the Fungal Cell. Berlin: Springer-Verlag. pp 225 241. Lora JM, Pintor-Toro JA, Benitez T, and Romero LC (1995). Qid3 protein links plant bimodular proteins with fungal hydrophobins. Mol Microbiol 18:380 382. Mahadevan MS, Amemiya C, Jansen G, Sabourin L, Baird S, Neville CE, Wormskamp N, Segers B, Batzer M, Lamerdin J, de Jong P, Wieringa B, and Korneluk RG (1993). Structure and genomic sequence of the myotonic dystrophy DM kinase gene. Hum Mol Genet 2:299 304. Moore RT (1985). The challenge of the dolipore/parenthosome septum. In: Moore D, Casselton LA, Wood DA, Frankland JC eds. Developmental Biology of Higher Fungi. Cambridge: Cambridge University Press. pp 175 212. Olsson S (2001). Colonial growth of fungi. In: Howard RJ, Gow NAR eds. The Mycota VIII. Biology of the Fungal Cell. Berlin: Springer-Verlag. pp 125 141. Plamann M, Minke PF, Tinsley JH, and Bruno KS (1994). Cytoplasmic dynein and actin-related protein Arp1 are required for normal nuclear distribution in lamentous fungi. J Cell Biol 127:139 149. Prokisch H, Yarden O, Mischler M, Tropschug M, and Barthelmess IB (1997). Impairing calcineurin of Neurospora crassa

reveals its essential role for hyphal growth, morphology and maintenance of the apical Ca2-gradient. Mol Gen Gene 256:104 114. Riquel M, Reyanaga-Pena CG, Gierz G, and Bartnicki-Garcia S (1998). What determines growth direction in fungal hyphae? Fungal Genet Biol 24:101 109. Ruiz-Herrera J (1992). Fungal Cell Wall: Structure, Synthesis and Assembly. Boca Raton: CRC Press. p 248. Saupe SJ, Kuldau GA, Smith ML, and Glass NL (1996). The product of the het-C heterokaryon incompatibility gene of Neurospora crassa has characteristics of a glycine-rich cell wall protein. Genetics 143:1589 1600. Seiler S, Plamann M, and Schliwa M (1999). Kinesin and dynein mutants provide novel insights into the roles of vesicle trafc during cell morphogenesis in Neurospora. Curr Biol 9:779 785. Sietsma JH, Beth Din A, Ziv V, Sjollema KA, and Yarden O (1996). The localization of chitin synthase in membranous vesicles (chitosomes) in Neurospora crassa. Microbiology 142:1591 1596. Steinberg G and Schliwa M (1993). Organelle movements in the wild type and wall-less fz;sg;os-1 mutants of Neurospora crassa are mediated by cytoplasmic microtubules. J Cell Sci 106:555 564. Steinberg G and Schliwa M (1995). The Neurospora organellar motor: a distant relative of conventional kinesin with unconventional properties. Mol Biol Cell 6:1605 1618. Steinberg G and Schliwa M (1996). Characterization of the biophysical properties of kinesin from the fungus Neurospora crassa. J Biol Chem 271:7516 7521. Strahl-Bolsinger S, Gentzch M, and Tanner W (1999). Protein O-mannosylation. Biochim Biophys Acta 1426:297 308. Sweigard JA and Ebbole DJ (2001). Functional analysis of pathogenicity genes in a genomics world. Curr Opin Microbiol 4:387 392. Talbot NJ (1999). Coming up for air and sporulation. Nature 398:295 296. Trinci APJ, Bocking S, Swift RJ, Withers JM, Robson GD, and Wiebe MG (1999). Growth, branching and enzyme production by lamentous fungi in submerged culture. In: Gow NAR, Robson GD, Gadd GM eds. The Fungal Colony. Cambridge: Cambridge University Press. pp 108 125. Turner G and Harris SD (1999). Genetic control of polarized growth and branching in lamentous fungi. In: Gow NAR, Robson GD, Gadd GM eds. The Fungal Colony. Cambridge: Cambridge University Press. pp 229 260. Verde F, Wiley DJ, and Nurse P (1998). Fission yeast orb6, a ser/thr protein kinase related to mammalian rho kinase and myotonic dystrophy kinase, is required for maintenance of cell polarity and coordinates cell morphogenesis with the cell cycle. Proc Natl Acad Sci USA 95:75206 75531. Wessels J (1993). Wall growth, protein excretion and morphogenesis in fungi. New Phytol 123:397 413. Wosten HAB, van Wetter MA, Lugones LG, van der Mei HC, Busscher HJ, and Wessels JGH (1999). How a fungus escapes the water and grows into the air. Curr Biol 9:85 88. Yamashita RA and May G (1998). Motoring along the hyphae: molecular motors and the fungal cytoskeleton. Curr Opin Cell Biol 10:74 79. Yarden O, Plamann M, Ebbole DJ, and Yanofsky C (1992). cot-1, a gene required for hyphal elongation in Neurospora crassa, encodes a protein kinase. EMBO J 11:2159 2166.

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